Pharmaceutical proteins are produced as recombinant proteins by expression in eukaryotic expression systems. After the synthesis of the protein backbone, the recombinant protein is submitted to further post-translational processing including the attachment of sugar residues, a process known as glycosylation. However, eukaryotic organisms exhibit different glycosylation processing involving specific enzymes (glycosyltransferases and glycosidases), and so that the glycosylation patterns, even of the same protein, will be different depending on the eukaryotic cell in which the particular protein is being produced. Thus, the glycosylation pattern of pharmaceutical proteins expressed in eukaryotic host cells differs substantially from the glycosylation pattern of the natural proteins produced in humans and other mammals.
N-Glycosylation: a Major Post-Translational Modification of Secreted Proteins
N-glycosylation is a major post-translational modification step in the synthesis of proteins in eukaryotes. N-glycan processing in the secretory pathway is essential for proteins intended to be secreted or integrated into membranes. N-glycosylation starts when the protein is translated and translocated from the ribosome into the lumen of the endoplasmic reticulum (ER). In this processing, a dolicholphosphate oligosaccharide precursor (Glc3Man9GlcNAc2-PP-dolichol) is initially assembled at the cytoplasmic face and finished in the luminal face of the ER membrane (BURDA AND AEBI, Biochimica et Biophysica Acta, vol. 1426, p: 239-257, 1999). This precursor is used by the oligosaccharyltransferase (OST) multisubunit complex that catalyses its transfer onto the asparagine residues of the consensus sequences Asn-X-Ser/Thr, when X is different than proline and aspartic acid, of a target protein (BURDA AND AEBI, above mentioned, 1999). The precursor is then deglucosylated/reglucosylated to ensure the quality control of the neosynthesised protein through the interaction with ER-resident chaperones calreticulin and calnexin. These ER events are crucial for proper folding and oligomerization of secreted proteins (HELENIUS AND AEBI, Science, vol. 291, p: 2364-2369, 2001), highly conserved in eukaryotes investigated so far. These steps lead to the formation of a limited set of high-mannose-type N-glycans (FIG. 1). In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a large variety of organism-specific complex structures. Mannosidases located in this compartment first degrade the oligosaccharide precursor into high-mannose-type N-glycans ranging from Man9GlcNAc2 (Man-9) to Man5GlcNAc2 (Man-5). N-acetylglucosaminyltransferase I (GnT I) then transfers a first GlcNAc residue on the β(1,3)-mannose arm of Man5GlcNAc2 and opens the door to the synthesis of multiple structurally different complex-type N-glycans (FIG. 1). Then, the actions of α-mannosidase II and GnT II allow the synthesis of the core GlcNAc2Man3GlcNAc2. The complex-type oligosaccharides arise from the transfer in the Golgi apparatus of monosaccharide residues onto the core GlcNAc2Man3GlcNAc2 under the action of organism-specific glycosyltransferases. As a consequence, mature proteins leaving the secretory pathway harbour multiple organism-specific complex N-glycans allowing the protein to acquire a set of glycan-mediated biological functions. As illustration, N-glycans in mammals are maturated into poly-antennary, poly-sialylated structures harbouring an α(1,6)-linked fucose residue on the proximal N-acetylglucosamine of the core (core-α(1,6)-fucose) (FIG. 1).
Remodelling into Human-Like N-Glycans by Knock-in Strategies
Since glycosylation profiles differs between mammals and eukaryotic host cells, strategies have been developed for the in vivo remodelling of the protein N-linked glycan structures. These strategies include the knock-out of endogenous genes that are involved in the transfer of some specific monomers, and knock-in methodologies based on the expression in the host cells of mammalian enzymes. The knock-in approach results, by complementing the enzyme repertoire of the host cell, in the synthesis in the recombinant expression system of N-linked glycans similar to those found in mammalian cells. As illustration, the remodelling of plant N-glycans into mammalian-like N-glycans has been achieved by expressing a human β(1,4)-galactosyltransferase in plant cells. Targeted insertion of the human β(1,4)-galactosyltransferase in Physcomitrella patens has also been carried out leading to the addition of terminal β(1,4)-galactose to endogenous N-glycans. Human N-acetylglucosaminyltransferase III (GnT III) has also been successfully expressed in plants in order to in planta engineer endogenous N-glycans. This transferase is able to introduce β(1,4)-GlcNAc residue on the β-mannose of the core mammalian N-glycans (bisecting GlcNAc).
With the exception of IgG, human serum proteins require sialic acid on terminal positions of their N-glycans (FIG. 1). Most non-mammalian eukaryotic cells, such as plants, do not synthesize N-acetyl- and N-glycolylneuraminic acids (Neu5Ac and Neu5Gc), the two main mammalian sialic acids. As consequence, genetic manipulation has been developed for the in planta synthesis of sialylated proteins by expressing enzymes able to synthesise CMP-Neu5Ac, its Golgi transporter and the appropriate sialyltransferases (Paccalet et al., 2007, Plant Biotechnology Journal, vol. 5, p 12-25; Castilho et al., 2008, Plant Physiol., vol. 147 (11), p 331-339; Castilho et al., 2010, The journal of Biological Chemistry, vol. 285 (21), p 15923-30.